CONTROL OF PHAGOCYTE NADPH OXIDASE BY CYTOSOLIC PROTEINS

胞浆蛋白对吞噬细胞 NADPH 氧化酶的控制

• 批准号: 6830822
• 负责人: GARY M BOKOCH
• 金额: $ 29.17万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6830822
• 关键词: NAD(P)H dehydrogenase; actin binding protein; biological signal transduction; chronic granulomatous disease; clinical research; cytoplasm; enzyme activity; enzyme biosynthesis; enzyme inhibitors; fluorescence microscopy; guanine nucleotide exchange factors; guanosinetriphosphatase activating protein; guanosinetriphosphatases; human subject; immunofluorescence technique; inflammation; laboratory rabbit; neutrophil; phagocytosis; vesicle /vacuole

DESCRIPTION (Applicant's Description Verbatim): The superoxide(02-)-generating NADPH oxidase of phagocytes is a potent source of reactive oxygen species (ROS). Its importance to innate immunity is manifested by the inherited syndrome chronic granulomatous disease, in which oxidase activity is absent and patients are susceptible to life-threatening microbial infections. O2-and its derivatives also cause severe tissue damage, contributing to inflammatory diseases such as ischemia-reperfusion injury and acute respiratory distress syndrome, and their prolonged generation in chronic inflammation can lead to dysplasia and malignancy. ROS produced by homologous systems in non-phagocytes are implicated in abnormal cell growth. The long-term objectives of the proposed research are to understand the mechanisms that control NADPH oxidase assembly and activity, and to identify steps that can be inhibited to minimize tissue damage. These four specific aims address gaps in knowledge that impede achievement of these goals. (1) Identify the processes of molecular recognition that encode the complex of cytosolic oxidase proteins to associate with the flavocytochrome to form a isolated phagosomes and cellular fractions, will be used to study the formation of the active enzyme and determine the roles of coronin and cofilin in controlling its assembly. 2) Analyze interactions between NADPH oxidase components, and identify factors that promote their association and dissociation. A biosensor instrument will be used to quantitate interactions between oxidase-related proteins and study how they are modified by changes that mimic intracellular signals. Development of this technique will provide an assay for molecules designed to inhibit oxidase activation. 3) Identify and isolate GTPase activating proteins and guanine nucleotide exchange factors for Rac2 and study their involvement in controlling Rac and NADPH oxidase activity. The identities of the regulatory proteins that control Rae activity in the neutrophil by altering its GDP/GTP-bound state are not known. These proteins are likely to be important in the negative and positive regulation of 02- generation. 4) Identify signaling events and molecules that terminate superoxide generation. The processes that switch off the enzyme have important clinical implications for inflammatory disease, but they are not well understood. Cell-free oxidase systems and intact cells will be used to determine if a) phosphoinositides; b) changes in the phosphorylation state of phox proteins; and c) changes in the GDP/GTP-bound state of Rae are negative regulators of the enzyme.

描述（申请人的逐字描述）：产生超氧化物(02-) 吞噬细胞的 NADPH 氧化酶是活性氧的有效来源 （ROS）。它对先天免疫的重要性通过遗传性表现出来。 慢性肉芽肿病综合征，其中氧化酶活性缺失并且 患者容易受到危及生命的微生物感染。 O2-及其 衍生物还会造成严重的组织损伤，导致炎症 缺血再灌注损伤和急性呼吸窘迫等疾病 综合征，以及它们在慢性炎症中的长期产生可能导致 发育不良和恶性肿瘤。非吞噬细胞中同源系统产生的 ROS 与细胞异常生长有关。该组织的长期目标 拟议的研究旨在了解控制 NADPH 氧化酶的机制 组装和活动，并确定可以抑制的步骤以尽量减少 组织损伤。这四个具体目标解决了阻碍 这些目标的实现。 (1) 识别分子识别过程 编码胞质氧化酶蛋白复合物以与 黄细胞色素形成分离的吞噬体和细胞组分，将 用于研究活性酶的形成并确定其作用 Coronin 和 cofilin 控制其组装。 2）分析交互作用 NADPH 氧化酶成分之间的关​​系，并确定促进其作用的因素 关联和解离。生物传感器仪器将用于定量 氧化酶相关蛋白之间的相互作用并研究它们是如何被修饰的 通过模仿细胞内信号的变化。这项技术的发展将 提供了一种设计用于抑制氧化酶激活的分子的测定方法。 3） 鉴定并分离 GTP 酶激活蛋白和鸟嘌呤核苷酸交换 Rac2 的影响因素并研究它们在控制 Rac 和 NADPH 中的作用 氧化酶活性。控制 Rae 的调节蛋白的特性 中性粒细胞通过改变 GDP/GTP 结合状态而产生的活性尚不清楚。 这些蛋白质可能在消极和积极的方面都很重要 02-代的调节。 4) 识别信号事件和分子 终止超氧化物的产生。关闭酶的过程有 对炎症性疾病具有重要的临床意义，但效果不佳 明白了。无细胞氧化酶系统和完整细胞将用于 确定是否a)磷酸肌醇； b) 磷酸化状态的变化 phox蛋白； c) Rae 的 GDP/GTP 结合状态的变化为负 酶的调节剂。